On the basis of long-term experience with the preservation of the pathogenic bacteria of the Czechoslovak National Collection of Type Cultures, it is concluded that the suspending medium is a factor of primary importance in the preservation of these organisms in the freeze-dried state. The primary index of suspending medium effectiveness is the level of protection it affords against the adverse effects of low (subzero) temperatures, dehydration, long-term storage, and rehydration. A number of simple and compound suspending media were studied for their protective effects on cultures from over 50 genera of predominantly pathogenic bacteria. The results indicate that not only each bacterial genus and species but also frequently every strain of a given serological type must be looked upon as a distinct biological entity, the preservation of the viability, activity, and desirable characters of which must be tested in different protective media in preliminary experiments. Heat testing (at 75 to 100 C) of freeze-dried cultures kept under high vacuum is recommended as a good indicator of adequate protection provided by a suspending medium. With the freeze-drying technique used, the best results for the largest number of species were obtained with a mixture of calf serum or defibrinated sheep blood and lactose solution (to a final concentration of 5 to 10%) and Annear's peptone-containing medium. Individual suspending media were evaluated in relation to individual strains, and recommendations are made as to the preferred medium on the basis of control tests performed at 5-, lo-, and 15-year intervals.Twenty-five years have elapsed since the establishment of the Czechoslovak National Collection of Type Cultures (CNCTC) at the Institute of Epidemiology and Microbiology (in 197 1, the Institute of Epidemiology and Microbiology became a part of the newly established Institute of Hygiene and Epidemiology under the name Center of Epidemiology and Microbiology). At present, the CNCTC contains over 7,000 strains of bacteria, viruses, and fungi, the majority of which have been successfully preserved by the relatively modern method of freeze-drying, first employed at the CNCTC in 1948. The freeze-drying technique has made important headway both in Czechoslovakia and abroad (16), the continuing refinement of this technique having proceeded along the lines of improvements in the freeze-drying apparatus (19) and in the suspending media used for freeze-drying ( 1 , 4 , 6 , 7, 9, 10, 12, 20, 2 1).A number of laboratories have recently summarized their long experience with freezedrying as applied t o the preservation of microorganisms (3,5,9,10,17) and by so doing have provided valuable information for culture collections and other laboratories interested in microorganism preservation. We present our experience on the preservation of the pathogenic bacteria maintained and stored at the CNCTC and of various recently isolated bacteria maintained at the National Reference Laboratories of the Institute of Hygiene and Epidemiology (22).
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